“Activation” of vitamin D by the liver

Ponchon, G.; Kennan, Alfred L.; DeLuca, Hector F.

doi:10.1172/jci106168

Cited by 363 publications

(136 citation statements)

References 35 publications

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“…To determine the maximum number of cDNAs that could be assayed at one time in the expression screen, dilution experiments were performed with the CYP27A1 expression plasmid, the receptor-reporter system, and the precursor substrate, 1␣-hydroxyvitamin D 3 . A luciferase enzyme activity that was 3-fold over background was observed when the sterol 27-hydroxylase plasmid was diluted 190-fold with a control plasmid lacking a cDNA insert.…”

Section: Resultsmentioning

confidence: 99%

“…On day 1, cells were transfected with a mixture containing 0.45-l FuGENE TM 6 and 150 ng of plasmid DNA comprising 20 ng of pCMX-␤-galactosidase (29), 20 ng of pTK-MH100X4-LUC (27), 20 ng of pCMX/GAL4-VDR (see below), 10 ng of pVA1 (30), 5 ng of pCMV6/mAdx, and 75 ng of cDNA pool. Cells were returned to the incubator for 8 -10 h, and then vehicle (ethanol) or 1␣-hydroxyvitamin D 3 Vitamin D Receptor Activation Assay and Ligands-The GAL4-VDR/ GAL4-luciferase receptor-reporter system was composed of the pTK-MH100X4-LUC plasmid (27), and pCMX/GAL4-VDR, which specifies a fusion protein consisting of amino acids 1-147 of the Saccharomyces cerevisiae galactose 4 (GAL4) transcription factor (encoded by nucleotides 2901-3353 of GenBank TM /EBI Data Bank accession no. X85976) and amino acids 90 -427 of the human vitamin D receptor (hVDR, encoded by nucleotides 383-1397 of GenBank TM /EBI Data Bank accession no.…”

Section: Methodsmentioning

confidence: 99%

“…Secosteroids used in the experiments reported here (vitamin D 2 , vitamin D 3 , 1␣-hydroxyvitamin D 3 , 25-hydroxyvitamin D 3 , and 1␣,25-dihydroxyvitamin D 3 ) were obtained from Sigma and added to the culture medium in ethanol (1 l/ml of medium). Stock solutions of these compounds ranged in concentration from 10 Ϫ2 to 5 ϫ 10 Ϫ7 M and were stored at Ϫ20°C.…”

Section: Methodsmentioning

confidence: 99%

“…Subsequently, the structure of vitamin D and its origins from plant and animal steroids were determined, and the roles of the vitamin in the mobilization of minerals from the diet and in bone were defined. The liver was shown to be required for the activation of vitamin D by 25-hydroxylation (2,3). Although 25-hydroxyvitamin D was more active than vitamin D in many bioassays (4), the most potent hormone was 1␣,25-dihydroxyvitamin D (5,6), which was synthesized from 25-hydroxyvitamin D in the kidney (7).…”

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confidence: 99%

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De-orphanization of Cytochrome P450 2R1

Cheng

Motola

Mangelsdorf

et al. 2003

Journal of Biological Chemistry

353

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Vitamin D regulates calcium and phosphate metabolism by activating the vitamin D receptor, a transcription factor and member of the nuclear receptor family. In the 1920s, McCollum, Mellanby, and Pappenheimer (see Ref. 1) showed that deficiency of vitamin D caused rickets in experimental animals. Subsequently, the structure of vitamin D and its origins from plant and animal steroids were determined, and the roles of the vitamin in the mobilization of minerals from the diet and in bone were defined. The liver was shown to be required for the activation of vitamin D by 25-hydroxylation (2, 3). Although 25-hydroxyvitamin D was more active than vitamin D in many bioassays (4), the most potent hormone was 1␣,25-dihydroxyvitamin D (5, 6), which was synthesized from 25-hydroxyvitamin D in the kidney (7). The molecular mechanism of vitamin D action was manifest with the identification (8) and cDNA cloning (9) of the vitamin D receptor.Hydroxylation reactions catalyzed by cytochrome P450s (CYP) 1 activate and inactivate vitamin D as a ligand for the receptor. 25-Hydroxylation is performed in the liver by two different enzymes, one located in the mitochondria (10), identified as the CYP27A1 sterol 27-hydroxylase (11-15), and a second in microsomes (16, 17), which has not been identified in most species. A second mitochondrial P450 (CYP27B1), for which an encoding cDNA was isolated by expression cloning (18) In the current study, a cDNA library made from hepatic mRNA of mice deficient in the gene encoding the mitochondrial CYP27A1 enzyme was screened using a vitamin D receptorbased, ligand activation assay (18). A single cDNA specifying a microsomal P450 enzyme termed CYP2R1 with previously unknown substrate specificity was identified. The biochemical properties and tissue distribution of CYP2R1 are consistent with this enzyme being the microsomal vitamin D 25-hydroxylase. EXPERIMENTAL PROCEDURESExpression Plasmids-A mouse adrenodoxin cDNA (mAdx, nucleotides 41-772 of GenBank TM /EBI Data Bank accession no. L29123) was amplified by the polymerase chain reaction (PCR) from random hexamer-primed mouse hepatic cDNAs using the following oligonucleotide

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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De-orphanization of Cytochrome P450 2R1

Cheng

Motola

Mangelsdorf

et al. 2003

Journal of Biological Chemistry

353

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“…25-OH-Ds was shown formed from vitamin Ds in intact rats but not to a signii&ant level in the hepatectomized rats [ 1,2]. This suggests that the transformation ofvitamin Da to 25-OH-Da takes place mainly, if not only, irt the liver ~ The tr~sfor~tion of &amin Da to 255H-Ds was demonstrated using both perfused rat liver and rat liver homogenate systems [31.…”

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confidence: 99%

In vitro 25‐hydroxylation of 1α,24‐dihydroxyvitamin D₃ in various rat tissues

et al. 1980

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The 25-hydroxylation of vitamin Ds in the liver is the first step for vitamin Ds activation [l-3]. 25-OH-Ds was shown formed from vitamin Ds in intact rats but not to a signii&ant level in the hepatectomized rats [ 1,2]. This suggests that the transformation ofvitamin Da to 25-OH-Da takes place mainly, if not only, irt the liver ~ The tr~sfor~tion of &amin Da to 255H-Ds was demonstrated using both perfused rat liver and rat liver homogenate systems [31. However, vitamin D&Lhydroxylase activity was reported present in the kidney and intestine as well as in the liver of chick [4], indicating the distribution of the enzyme in various tissues.We have shown that lar,24(OHhDs is metabolized rapidly to lcw,24,25-(OH)sDa in rats [5,6] and the 25" hydroxylation occurs mainly in the liver [S]. However, the possibility of 25hydroxylation in the tissues other than the liver has been suggested also, since a small amount of laJ4,25-(OH)sDs was formed from la;24fOffaDs in the hepatecto~d rats. This paper describes the 25-hydroxylation of la@&Xl)sDs and la-OH-Da using homogenates of rat liver, kidney, intestine, bone, adrenal and muscle. Wear&rig male Wistar rats were fed a vitamin D-deficient low calcium diet for 6 weeks f5]. 10% liver homogenates in 0.25 M sucrose were prepared with the aid of a Potter-Elvehjem homogenizer fitted with a teflon pestle. A portion (5 ml) of the homogenates was then mixed with 10 ml phosphate buffer (pH 7.4) containing Mg-and NADPH-generating systems as in [3]. The final concentrations were: 25 mM phosphate buffer (PH 7.4), 1.25 mM MgCls, 25 mM KCl, 5 mM ATP, 0.1 mM NADP, 40 mM nicotinamide, 5 mM sodium succinate, 5.6 mM glucose 6-phosphate and 09 units glucose 6-phosphate dehydrogenase. The reaction was initiated by adding 1 .O @g ~#~qR~~O~~ f3HlDa, 1~,2q~~O~ fsHlDa or ia-OH-j3H]Ds in 200 ~1 ethanol. The incubation was carried out with gentle shaking at 37°C for 2 h and 30 ml methanol:chloroform (1: 1) was added to stop the reaction. Incubation of intestinal mucos~ homogenatesIntestines from the vitamin D-deficient rats were rinsed with 0.9% NaQ solution, then the mucosa was scraped off from the serosa with a glass microscope slide. The intestinal mucosa (3 g) was homogenized with 30 ml 0.25 M sucrose in a Potter-Elvehj~ homoge~zer equipped with a Teflon pestle, The incubation was carried out with the same reactian mixture as for the liver homogenate incubations. The 149

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